Project Summary/Abstract The gut microbiota has been linked to many features of human health, spawning efforts to develop microbiota-directed therapeutics or prebiotics. Identifying strategies/reagents for safe, efficacious manipulation of the microbiota is a high priority goal of current precision medicine initiatives. Achieving this goal requires informative preclinical models to describe the magnitude and mechanism of prebiotics? effects on gut microbes and host biology, and to obtain a fundamental understanding of gut community dynamics. Fiber-based foods have been shown to have beneficial microbially-mediated effects on health. Understanding how dietary fibers produce these effects is confounded by (i) their compositional complexity (e.g., what are the bioactive glycans?), (ii) limited knowledge of which bacteria use specific constituent glycans in fiber, and (iii) competition and cooperation between bacteria over these glycans. I hypothesize that the polysaccharide components of fibers and gut community membership interact to dictate responses to fiber-based prebiotics. Knowledge of these interactions, and their underlying mechanisms, will inform development of improved microbiota-directed therapeutics. I plan to test this hypothesis in a series of experimental aims. AIM 1 will identify fiber-dependent bacterial interactions in a model human gut microbiota composed of sequenced, cultured bacterial strains installed in gnotobiotic mice. I will systematically omit bacteria from this model community prior to its introduction into mice fed a representative ?unhealthy? low fiber high saturated fat USA diet supplementation with a purified plant-derived dietary fiber. I will analyze the effects of community manipulation/fiber supplementation on bacterial gene expression and abundance. Using forward genetic screens (whole genome transposon mutant libraries) and mass spectrometry-based proteomics and metabolomics, I will characterize the genetic determinants of bacterial fiber responses and the bioactive components of fiber preparations across informative community contexts. AIM 2 will extend these gnotobiotic/multi-omic studies through a novel use of CRISPR technology that allows for selective depletion of targeted bacteria from an established community in a fiber-supplemented diet context. This work will refine our understanding of microbiota function and how we might drive a community toward stable, beneficial states. I will generate and analyze many multi-omic datasets while receiving excellent scientific training from leading researchers in human microbiome science and computational biology. Together, with the stellar clinical mentorship and training provided by Washington University SOM, this proposal will help me to develop into an independent physician-scientist.